Insight into post-transcriptional regulatory mechanisms will be sought through the study of a novel paradigm in global regulation, the carbon storage regulatory (Csr) system of Escherichia coli. Csr includes CsrA, an RNA binding protein that regulates translation and/or modulates the stability of target mRNAs. CsrB and CsrC are non-coding regulatory RNAs that antagonize CsrA by sequestering this protein and CsrD is a protein that specifically targets CsrB and CsrC for degradation by RNase E. In E. coli, CsrA affects metabolism, physiology, motility and multicellular behavior on a broad scale, repressing certain genes expressed during the transition from exponential to stationary phase growth and activating various genes expressed during exponential phase. CsrA homologues are widely distributed among eubacteria and regulate the expression of virulence factors in both plant and animal pathogens. Thus, the proposed studies will also provide fundamental understanding of the regulation of bacterial physiology and pathogenesis, and may suggest novel therapeutic approaches for bacterial infections. The specifc aims of this proposal are: 1) Elucidate the molecular mechanisms by which CsrA activates or inhibits gene expression. This aim will include an analysis of CsrA-mediated autoregulation, as well as an investigation into the factors that influence the stability of CsrA target transcripts. 2) Establish the Csr global regulon using a combination of bioinformatic, genomic, molecular genetic and biochemical approaches. 3) Stoichiometric and structural characterization of CsrA-RNA complexes. This aim will include NMR structural determination of a model CsrA target RNA, as well as the structure of CsrA-RNA complexes by X-ray crystallography. 4) Elucidate the molecular mechanism of CsrD action. We will conduct experiments to elucidate the mechanism by which CsrD specifically targets CsrB and CsrC for degradation by RNase E. The long-range objectives of these studies are to fully understand the regulatory components, genetic circuitry, molecular mechanisms, and biological functions of the Csr system. Insight into post-transcriptional regulatory mechanisms will be sought through the study of a novel paradigm in global genetic regulation, the carbon storage regulatory (Csr) system of Escherichia coli. Csr controls bacterial metabolism, physiology, motility and biofilm development on a broad scale, and regulates the expression of virulence factors in both plant and animal pathogens. Thus, the proposed studies will provide fundamental understanding of the regulation of bacterial physiology and pathogenesis, and may suggest novel therapeutic approaches for bacterial infections.